Heat-shock protein 90 (Hsp90) coordinates the trafficking and regulation of diverse signalling proteins, but its precise role in regulating specific cellular targets is not known. Here we show that Hsp90 associates with endothelial nitric oxide synthase (eNOS) and is rapidly recruited to the eNOS complex by agonists that stimulate production of nitric oxide, namely vascular endothelial growth factor, histamine and fluid shear stress. Moreover, the binding of Hsp90 to eNOS enhances the activation of eNOS. Inhibition of signalling through Hsp90 attenuates both agonist-stimulated production of nitric oxide and endothelium-dependent relaxation of isolated blood vessels. Our results indicate that Hsp90 facilitates signalling mediated by growth-factor, G-protein and mechanotransduction pathways that lead to the activation of eNOS. These observations indicate that in addition to its role as a molecular chaperone involved in protein folding and maturation, Hsp90 may also be recruited to cellular targets depending on the activation state of the cell.
The regulation of endothelial nitric oxide synthase (eNOS) by phosphorylation is poorly understood. Here, we demonstrate that eNOS is tyrosine-phosphorylated in bovine aortic endothelial cells (BAEC) using 32 P metabolic labeling followed by phosphoamino acid analysis and by phosphotyrosine specific Western blotting. Treatment of BAEC with hydrogen peroxide and the protein tyrosine phosphatase inhibitor, sodium orthovanadate, increases eNOS tyrosine phosphorylation. Utilizing a novel immunoNOS assay, the increase in tyrosine phosphorylation is associated with a 50% decrease in the specific activity of the enzyme. Because eNOS is localized in plasmalemma caveolae, we examined if tyrosine phosphorylated eNOS interacts with caveolin-1, the coat protein of caveolae. Immunoprecipitation of eNOS from bovine lung microvascular endothelial cells resulted in the co-precipitation of caveolin-1. Conversely, immunoprecipitation of caveolin-1 resulted in the co-precipitation of tyrosine-phosphorylated eNOS. Thus, tyrosine phosphorylation is a novel regulatory mechanism for eNOS and caveolin-1 is the first eNOSassociated protein. Collectively, these observations provide a novel regulatory mechanism for eNOS and suggest that tyrosine phosphorylation may influence its activity, subcellular trafficking, and interaction with other caveolin-interacting proteins in caveolae.Post-translational modification of proteins is a widely used mechanism for transmitting biological signals and regulating the activities, biosynthesis, and degradation of enzymes. One of these modifications, protein phosphorylation, plays a central role in signal transduction pathways regulating many biological processes, including cellular proliferation, migration, and differentiation. More specifically, phosphorylation of tyrosine residues is an essential step in cellular activation by many external signals, including growth factors, cytokines, and cellular stress (1, 2).In the cardiovascular and nervous systems, activation of cell surface receptors triggers the immediate synthesis of nitric oxide (NO).1 In the cardiovascular system, NO is derived from one of the chemically equivalent guanidino nitrogens of L-arginine, in a reaction catalyzed by endothelial NO synthase (eNOS or NOS 3). In intact blood vessels and in cultured endothelial cells (EC), eNOS is rapidly activated by agonists that mobilize intracellular calcium. Increases in intracellular calcium facilitate interactions with calmodulin and the activated calciumcalmodulin complex can stimulate NADPH-dependent electron flux through eNOS to produce NO (3). Recent evidence suggests that eNOS can also be activated in a calcium-independent manner by fluid shear stress (4 -6) and insulin-like growth factor (7), presumably mediated through a tyrosine kinase cascade, since inhibitors of tyrosine kinases, but not chelation of intracellular calcium, inhibits NO release. Whether eNOS is directly tyrosine-phosphorylated or indirectly linked to a tyrosine kinase cascade is not known. Once NO is produced by either ...
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